Agent for treating illnesses of the tracheobronchial tract, especially chronic obstructive pulmonary disease (COPD)

ABSTRACT

The invention describes a means for the treatment of diseases of the tracheo-bronchial tract, particularly of COPD.

RELATED APPLICATIONS

This application is a continuation of PCT patent application Ser. No.PCT/EP01/02832, filed Mar. 13, 2001, which claims priority to Germanpatent application number 10012151.9, filed Mar. 13, 2000, thedisclosures of each of which are incorporated herein by reference intheir entirety.

TECHNICAL FIELD

The invention relates to a means for the prophylaxis and therapy ofdisorders of the tracheo-bronchial tract, particularly of COPD (chronicobstructive pulmonary disease).

BACKGROUND ART

Typically, COPD is encountered in smokers. The disease is characterizedby chronic inflammation of the small airways (<2 mm) which unavoidablyresults in tissue reconstruction and irreparable narrowing (obstruction)of this portion of the airways.

Detailed tissue examination of these small airways shows increasedproduction of mucus, increase in muciferous goblet cells, smooth musclehypertrophy as well as infiltration with pigment-charged macrophages andCD8 positive T lymphocytes. As this process continues, tissuereconstruction and an increased deposition of connective tissue fibersoccur.

Pathophysiologically, it is the inflammation which is most important. Itis characterized by an infiltration of the small airways tissue withneutrophile granulocytes, macrophages, and lymphocytes. Thisinfiltration into the tissue is facilitated by the production ofproteases by leucocytes. Mediators are produced within the tissue whichpromote mucus formation, stimulate muscle cells and support thegeneration of connective tissue (collagen) fibers by fibroblasts.

In examinations of the respiratory mechanic, COPD shows a decreasedmaximal expiratory flow (FEV₁) and a slow forced emptying of the lungs.COPD is often associated with chronic bronchitis and emphysema. However,these two diseases can be clearly distinguished from COPD.

Clinically, chronic bronchitis is defined by a chronic productive coughfor at least 3 months per year in at least 2 successive years. Thisdisease is characterized by common bacterial infections and it can butdoes not necessarily lead to an irreversible obstruction.

Emphysema is defined as an irreversible dilatation of the airways distalof the terminal bronchia caused by degradation of the alveolar septsassociated with a degradation of elastic fibers. Emphysema is notaccompanied by fibrosis.

Thus, COPD may be differentiated as an entity from chronic bronchitisand emphysema although COPD may be accompanied by these two diseases.

Predominantly, COPD affects the small airways while chronic bronchitisoccurs in the large and medium airways and emphysema in the alveoles.

COPD is an irreversible process resulting in tissue rearrangementsaccompanied by a fibrosis (an increase in fibers). Chronic bronchitisdoes not result in tissue rearrangements. In emphysema, destruction ofthe alveolar septs, is observed.

An increase in neutrophile granulocytes, macrophages, and CD8 cells inthe small airways has been found to be typical in COPD. All inflammatorycells are involved in chronic bronchitis while leucocytes are of nomajor importance in emphysema.

It is known from EP 0 352 412 to use esters of retinoic acid and/or anester of retinol in the preparation form of an aerosol inhalant fortopical application by inhalation to prevent and treat mucous membranedisorders of the tracheo-bronchial tract in humans and animals. Theseso-called bronchitides include acute and chronic bronchitides which showno obstruction. For example, the majority of smokers suffers fromchronic bronchitis without showing airway obstruction and thus COPD.Bronchiectases are irreversible, cylindric, saccular or varicoiddilatations of the bronchia, a syndrome not belonging to the chronicobstructive disease, COPD.

Therefore, the mucous membrane disorders of the tracheo-bronchial tractmentioned in EP 0 352 412 do not belong to the diseases to be subsumedunder the term COPD. They differ from COPD particularly in that they donot show the irreversible obstruction typical for COPD.

Up to now, COPD has been treated for example by administration ofβ-adrenergics/anticholinergics, theophyllin, and/or glucocorticoids.Disadvantages of the above-mentioned medicaments are that their activityis only symptomatic and that they have no effect on the fatal course ofCOPD. Spasmolytics have a life shortening effect. Theophyllin causesarrhytmias. An important side effect of glucocorticoids is osteoporosis.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel means forthe treatment of COPD which avoids the disadvantages known from theprior art.

According to the invention, this object has been achieved by usingvitamin A and/or the derivatives and/or the esters thereof in atherapeutically effective amount in the therapy of COPD applied in theform of an aerosol inhalant.

The term “derivatives” of vitamin A comprises retinol, retinal, andretinoic acid and the derivatives and esters of each of these compoundsas well as β-carotene and its derivatives being the precursor of vitaminA. For example, retinol may be esterified with saturated and unsaturatedfatty acids such as linoleic acid, stearic acid, palmitic acid, oleicacid. This includes also chemical modifications of the fatty acids andalcohols mentioned. (See also EP 0 352 412 B1; page 11, claim 4+claim6). Martindale, The Extra Pharmacopeia, 1982, London, The PharmacueticalPress, and all subsequent editions is mentioned as an example. Thisdocument is incorporated herein by reference in its entirety. Foradministration by inhalation, vitamin A is applied in the form of anaerosol wherein particularly sprays and inhalants are used. Thisachieves the dispersion of the active agent into very small particles totransport it to the site of its therapeutic effectiveness.

According to the invention, vitamin A and the derivatives and estersthereof are present in the form of an aerosol. Aerosols are fine mistsusually obtained by systems under pressure. They may be obtained forexample by spraying or atomizing liquids but also of dry powders.Aerosols for inhalation are different from those systems usuallyreferred to as aerosols in that the aerosol particles are present in asize lower than 10 μm, preferably in the range of 1–10 μm.

The essential components of aerosol systems in the form of sprays arefor example a container containing a propellant while today also aerosolsystems without propellant have been developed. Furthermore, the aerosolsystem contains the active ingredient(s) together with conventionalauxiliary agents such as for example propellants and lactose. Thecomposition of these components determines the properties of the aerosolinhalant, i.e. for example the particle size distribution, deliveryrate, viscosity, etc.

Aerosols may be obtained as two-phase aerosols (gas and liquid) orthree-phase aerosols (gas, liquid, and solid or liquid). Two-phaseaerosols consist of a solution of the active agent in a liquefiedpropellant and of the atomized propellant. The solvent for examplecomprises the propellant or a mixture of the propellant and co-solventssuch as alcohol, propylene glycol, and polyethylene glycols often usedto ameliorate the solubility of the active agents.

Three-phase systems comprise a suspension or emulsion of the activeingredient or ingredients, respectively, together with the atomizedpropellants. A suspension includes the active agent dispersed in thepropellant system using conventional auxiliary agents such as wettingagents and/or solid carriers such as talc or colloidal silica.Propellants are known and comprise several carbohydrates, if possiblethose without damaging impact on the ozone layer.

An inhalation is only possible if the solutions for inhalation areatomized into very small particles of a size in the range of 1–10 μm.Only those finely dispersed aerosols can be inhaled at all. Aninhalation is contra-indicated in the case of conventional aerosols. Forfurther information with respect to aerosol inhalants see for examplethe US Pharmacopoeia which is incorporated herein by reference in itsentirety.

In a preferred embodiment of the invention, vitamin A is encapsulated inliposomes. Liposomes are aqueous compartments surrounded by a completelyclosed lipid bilayer. Liposomes are for example generated artificiallyby suspending suitable lipids in aqueous solutions. The liposomes aredispersed to generate closed vesicles of an approximately equal size.Into the aqueous solutions and the lipid membranes, respectively, maythen be incorporated foreign molecules, vitamin A according to thepresent invention. It has been shown that vitamin A packaged intoliposomes can enhance the therapeutic effect and efficiently reduce sideeffects. Further details in this respect are explained in the following.

It has been shown that liposomes containing at least phosphatidylcholinetogether with at least another phospholipid selected fromphosphatidylinositol, phosphatidylserine, phosphatidic acid andcardiolipin are particularly suitable for the treatment of COPD. The useof these specifically composed liposomes results in selective uptake byalveolar macrophages, a higher specificity of the therapy and areduction of the therapeutically effective dose in association withreduced side effects.

The amount of phosphatidylcholine is approx. 50–95% while the amount ofthe other phospholipids is 5–50%, each based on 100% phospholipids.Thus, phosphatidylcholine is an essential component of the liposomeswhile the remainder, i.e. the other 5–50%, may be one or more of theabove-mentioned phospholipids. The liposomes of the present inventionfurther contain antioxidants in an amount of 10–50%. Preferred in thisrespect is vitamin E or the derivatives thereof such as (+)-α-tocopherolacetate, (±)-α-tocopherol acetate, (±)-α-tocopherol acid succinate. Theamount of phospholipids is 50%–90%.

The amount of vitamin A encapsulated into the liposomes may bedetermined by those skilled in the art by conventionalmedical-therapeutic testing using the pure substance. It should beunderstood that these tests have to be carried out in animalsbeforehand. It is sensible to use also the liposome formulationdescribed as an aerosol content to achieve optimal therapeutic effects.

To date, vitamin A preparations have not been described as aerosolinhalants in the form of the specifically composed liposomes describedherein. A pharmaceutical preparation of this type may not only be usedin the treatment of COPD but e.g. also for the treatment of chronicbronchitides, emphysemas and malignant as well as benign lung diseasesand generally in the treatment of disorders of the tracheo-bronchialtract.

The liposomes used according to the invention differ from themultilamellar liposomes known from the prior art not only in theircomposition but also in that they are obtained in an unilamellar state.The specific combination of liposome organization, active agent, andstructure results in the surprising efficacy found according to thepresent invention in the treatment of diseases of the tracheo-bronchialtract.

Preferably, such phospholipids are employed which enable a selectivetargeting of alveolar macrophages by binding to specific receptorspresent on alveolar macrophages, the so-called scavenger receptors. Thisincludes the following phospholipids:

1. Phosphatidylinositol(1,2-diacyl-sn-glycero-3-phospho-[1-D-myo-inositol])

The acyl residues may be derived from arachidonic acid or from linoleicacid, palmitic acid, stearic acid and other unsaturated or saturatedfatty acids

2. Phosphatidylserine (1,2-diacyl-sn-glycero-3-phospho-serine)

The acyl residues may be derived from arachidonic acid or from oleicacid, palmitic acid and other unsaturated and saturated fatty acids

3. Phosphatidic Acid (1,2-diacyl-sn-glycero-3-phosphate)

The acyl residues may be derived from arachidonic acid or from stearicacid, decanoic acid, heptadecanoic acid, lauric acid, myristic acid,octanoic acid, oleic acid, palmitic acid, and other unsaturated orsaturated fatty acids

4. Cardiolipin (Diphosphatidylglycerol)

Cardiolipin contains a maximum of 4 acyl residues derived fromunsaturated and saturated fatty acids

5. Phosphatidycholine (L-α-lecithine,1,2-diacyl-sn-glycero-3-phosphocholine) (Partially Oxidized)

The acyl residues may be derived from arachidonic acid or fromunsaturated or saturated fatty acids. At least 1 acylic ligand isunsaturated. The unsaturated acyl residue is partially oxidized andmediates the binding of the liposomes to scavenger receptors.

The liposomes employed according to the present invention preferablyhave a size in the range of 0.1 μm to 2 μm, particularly preferred of0.4 μm. The use of unilamellar liposomes is particularly preferred.Their preparation is carried out by a type of high pressure filtration,for example using an extruder commercially available from LipexBiomembranes company, Canada. Conventional liposomes such as thosedescribed for example in the article by Parthasarathy et al., CancerChemother, and Pharmacol. 1999, 43(4), 277–283, are prepared byreconstitution of a dry lipid film in the desired liquid. This resultsin so-called multilamellar liposomes, i.e. liposomes having severallipid layers surrounding the aqueous core. Such liposomes are quiteheterogenous in size and may be brought to uniform size by means ofsonication, as shown in Parthasarathy et al., after which, however, theystill remain multilamellar. However, the multilamellar liposomesdescribed in Parthasarathy et al. tend to form aggregates or fuse toform still larger liposomes. The use of such liposomes has been foundunsuitable according to the invention. By using the extruder describedabove and has been mentioned exemplarily only the finished reconstitutedliposomes may be washed prior to use so that substances which have notbeen incorporated are removed, on the other hand the unilamellarliposomes having only one lipid layer around the aqueous core arefurther processed which renders them particularly stable. It is thecombination of the lipid composition described herein and the use ofunilamellar liposomes which leads to a particularly significanttherapeutic efficiency.

The object, namely to deliver the active agent to specific target cells,i.e. alveolar macrophages, can only be achieved by using the liposomepreparation provided according to the invention. Only this specificcomposition of the liposomes ensures that these are recognized andactively taken up by specific target cells. In this manner it ispossible to achieve an effect of vitamin A in alveolar macrophages asthe relevant target cells which has neither been known nor described upto now.

The use of the pharmaceutical composition according to the inventionresults in a very specific modification of the metabolism of a certaintype of leucocytes in the lung, namely the alveolar macrophages, tostrongly affect the inflammatory process, particularly of COPD.

It has not been known from the prior art that cellular targeting can beachieved by using a particular liposomal composition to bring aboutnovel therapeutic possibilities. Cellular targeting of the liposomecomposition selected according to the invention is the prerequisite forthe solution of the problem underlying the present invention in other,i.e. not-alveolar macrophages, another effect of vitamin A may beconsidered which could even compromise the above-mentioned object. Suchan effect has been described for example to occur in rat osteoblasts.

In Kirilenko V. N. and Gregoriadis G., J. Drug Targeting 1993, 1(4),361–368 lipids are described as emulsifiers enhancing the unspecificresorption of vitamins during food intake. The particular compositionused according to the invention is not described, and the use for atreatment of diseases of the tracheo-bronchial tract, particularly ofCOPD, is not suggested. Only the specific composition used hereinprovides the possibility to transport vitamin A to specific target cellsby which it can be actively incorporated.

In EP-A-0 229 561 liposomes are used as carriers to transport vitamin Ato skin cells. The composition used exclusively shows cosmetic but notherapeutic effects. It is unthinkable to use these compositions in thetracheo-bronchial tract. The liposomes mentioned in EP-A-0 229 561 alsolack the property of recognizing a particular target cell in the lung tobe incorporated by this cell in a highly specific manner.

In addition, also EP-A-0 352 412 is not prejudicial as to the novelty ofand does not render obvious the object of the present invention.Although it describes the treatment of the bronchial epithelium byvitamin A it does not use the specific liposomal preparation accordingto the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following the invention will be described with respect to theaccompanying Figures. The Figures show:

FIG. 1: BAL cells+/−vitamin A liposomes—patient E.S.: MMP-9 day 3;

FIG. 2: BAL cells+/−vitamin A liposomes—patient E.S.: TIMP-1 day 3;

FIG. 3: BAL cells+/−vitamin A liposomes—ratio MMP-9/TIMP-1: patient E.S.day 3;

FIG. 4: TNF ELISA of BAL cells patient: F. (sclerotic process);

FIG. 5: TNF ELISA of BAL cells patient: E. (sclerotic process);

FIG. 6: TNF ELISA of BAL cells patient: M. (EAA—farmer's lung);

FIG. 7: TNF ELISA of BAL cells patient: A. (giant cell pneumonia);

BAL=bronchio-alveolar lavage

TNF=tumor necrosis factor

AM=alveolar macrophages

ELISA=enzyme linked immunosorbent assay

In the following, the present invention will be described in more detailwith respect to Examples to which the invention, however, is notlimited.

DETAILED DESCRIPTION OF THE INVENTION

There is increasing evidence that disorders in the protease/proteaseinhibitor equilibrium are the cause underlying the pathogenesis ofchronic lung diseases. Thus, it has been demonstrated that matrixmetalloproteases (MMPs) secreted by alveolar macrophages lead toirreversible damages of the lung tissue and thus result in a decrease ofrespiratory functions. This is particularly true for patients sufferingfrom chronic obstructive bronchitis (COPD). Among the MMPs in COPD, itis particularly the secretion of MMP-9 metalloprotease which isincreased in the airways and alveoles and can no longer be completelyneutralized by its natural antagonist TIMP-1 (tissue inhibitor ofmetalloprotease). The alveolar macrophage has been suggested to be themain producer of MMP-9 in the lung and it (and not the neutrophilegranulocyte as supposed up to now) has also been discussed as the mainresponsible for the formation of emphysemas in most recent studies inrat. Our own studies show that MMP-9 is also increased in the serum ofCOPD patients. An important criterion to determine the degree of thedisturbance of the protease/antiprotease equilibrium is theproteinase-to-inhibitor ratio: MMP-9/TIMP-1.

Using an in vitro system with alveolar macrophages it has been shownaccording to the invention that vitamin A not only significantlyinhibits MMP-9 expression but surprisingly at the same time alsosignificantly stimulates the expression of the specific inhibitorTIMP-1. These effects were most pronounced if vitamin A was previouslypackaged into the specifically composed liposomes described above whichensures the specific uptake by alveolar macrophages.

Unexpectedly, by using liposome-encapsulated vitamin A the ratio ofMMP-9/TIMP-1 could be clearly reduced, i.e. by a factor of 12. Thismeans an efficient reduction of the tissue destructive potential of theproteolytic system by vitamin A. On the basis of these studies thepresent therapy approach for the treatment of COPD and lung emphysemahas been established. The composition of the liposomes is alreadydescribed above. The following Examples explain in detail thepreparation of the liposomes.

The effect of vitamin A on metalloproteases is known. However, theresults achieved by the administration of vitamin A are verycontradictory. While in several test systems there has been reported areduction in metalloprotease concentration by inhibition of theexpression of the respective coding genes, others describe an increasedexpression and thereby an increased concentration of metalloproteases.Park and Kim, Mol. Cells 1999, 9(2):119–26 describe that due to theeffect of retinoic acid enzymes having gelatinase activities which areto be counted among the metalloproteases were increased in theiractivity, particularly the expression of these metalloproteases wasincreased. The publication of Overall, Journal of Cellular Physiology,1995, 164(1):17–25 reports that retinoic acid increases the expressionof metalloproteases in rat osteoblasts while simultaneously the mRNAlevels of TMP-1 were clearly reduced depending on the dose of retinoicacid administrated. The work of Heath et al., Biochemical andBiophysical Research Communications 1990, 168(3):1171–1176 describesthat the TIMP levels decreased to 0 under the influence of retinol.

Considering the above-described state of the art it is a surprisingresult for those skilled in the art that by the effect of vitamin A,i.e. retinoic acid and retinol, not only a clear inhibition of theexpression of MMP-9 metalloprotease in alveolar macrophages and bloodmonocytes but simultaneously also a stimulation of the TIMP-1 inhibitoris achieved.

In the frame of experiments to determine the uptake of liposomesaccording to the invention by monocytes in blood there were used as testsystems also various conventional cell lines of different origin andexamined with respect to their capability to take up liposomes.Particularly, cells of myelomonocytic origin were used such as the celllines HL-60, U937, THP-1, and Mono Mac 6 (mentioned in the order ofincreasing degree of maturity). Thus, the monocytic human cell line MonoMac 6 has reached the highest degree of maturity on its way to developinto a macrophage. However, this cell has taken up almost no liposomeswhich is in contrast to the assumption that a cell with a higher degreeof differentiation also shows a higher phosphatidylserine receptorexpression. Insofar it was also not obvious that alveolar macrophageswould take up these liposomes in a selective manner. Furthermore, it wasunpredictable into which type of macrophage a blood monocyte woulddevelop since there are significant differences not only with respect tomorphology and function. Moreover, it was unknown which cells areendowed with what kind of phosphatidylserine receptor. Particularly, itwas unknown that a phosphatidylserine receptor is present on alveolarmacrophages. For this reason, not only the effect of vitamin A in thetreatment of COPD and lung emphysema was surprising but also theincreased efficacy of vitamin A due to its administration in the form ofliposomes, particularly by specifically composed liposomes as presentlydescribed.

By treatment of COPD and lung emphysema with vitamin A the imbalancebetween MMPs and TIMPs may be treated, i.e. the expression of the formeris inhibited while the expression of the latter is stimulated.

The therapy described herein counteracts tissue destruction and providesa novel and promising approach for the treatment of chronic obstructivelung disorders. Among others, the particular advantages of the inventionare:

-   -   use of a smaller dose due to selective uptake of liposomally        packaged vitamin A by the alveolar macrophages which are the        most important source of metalloproteases;    -   a highly efficient effect of vitamin A is achieved by the        simultaneous impact on two agonists in equilibrium of the        proteolytic system of the lung;    -   the protease/antiprotease equilibrium in the lung is returned to        normal;    -   liposomally packaged vitamin A is stored in the form of a depot;    -   his results in a lower amount of vitamin A released into the        blood circulation, the active agent remains directly at the site        of activity, i.e. the lung, and the systemic side effects are        decreased.

The following experiments demonstrate the surprising effects of vitaminA administration, particularly in the form of liposomes. For thispurpose, human alveolar macrophages (AMs) were obtained via bronchiallavage. The AMs were seeded into Mono Mac 6 medium+10% FCS onto a 24well “low attachment” plate (Costar) using 1×10⁶ cells in a volume of 1ml per each sample. Additions of vitamin A liposomes or of the puresubstances, respectively, were carried out. Cells for RT-PCR or culturesupernatant for protein determinations by means of ELISA, respectively,were harvested from parallel samples each on day 1, day 3, and day 4.Specifically, the following samples were prepared:

-   -   β=control sample, AMs in medium only    -   Lipleer=empty liposomes    -   LipVitA=liposomes containing vitamin A (retinoic acid, Sigma        R-2625) 5 μM final    -   VitA=pure vitamin A (retinoic acid, Sigma R-2625) 5 μM final    -   LipPalm=liposomes containing all-trans retinol palmitate (Sigma        R-3375) 3 μM final    -   Palmitat=all-trans retinol palmitate (Sigma R-3375) 3 μM final

The culture supernatants were monitored for MMP-9 and TMP-1 contentusing commercial ELISA kits (Amersham Pharmacia). In each case thenumerals refer to ng/ml culture supernatant based on 1×10⁶ cells.

The data given show the results obtained on day 3 following vitamin Aaddition. On day 1, no effect could be observed on the protein level(but on the RNA level); the data obtained on day 4 were the same asthose of day 3: while due to liposomal and free vitamin A the MMP-9expression is strongly reduced a simultaneous increase in TIMP-1 isobserved. In each case the effect of liposomal vitamin A is superior tothat of free vitamin A. This opposite regulation of MMP-9 and TIMP-1also explains the drastic effect on the MMP-9/TIMP-1 ratio of thevitamin A treatment.

The culture of BAL cells and PBMCs with liposomal vitamin A is performedas follows:

Material:

-   -   RPMI 1640 (Biochrom # F1415, Berlin), L-glutamine 2 mM (Gibco #        25030-024), penicillin 200 U/ml, streptomycin 200 g/ml (Gibco #        15140-114), non-essential amino acids (NEAA) 1-2×(Gibco #        11140-35), OPI supplement (contains oxalacetate, Na pyruvate and        insulin) 10 ml per 1 liter (Sigma # 0-5003). Following        filtration through a Gabro ultrafilter U 2000 (Martinsried,        Germany) to remove LPS addition of 10% FCS, LPS tested.    -   Costar low attachment 24 well plates # 3473 (Bodenheim, Germany)

1×10⁶ BAL cells (=cells obtained by brocho-alveolar lavage containingalveolar macrophages in an amount of about 80%) or 1.5×10⁶ PBMCs(=peripheral blood mononuclear cells isolated via LymphoPrep gradient(Nycomed, Norway)), respectively, in a volume of 1 ml were used persample. Pretreat the 24 well low attachment cell culture plate accordingto the manufacturer's instructions. For this purpose, fill therespective wells with 1 ml of medium and incubate for 20 min at 37° C.in an incubator. Afterwards remove the medium and replace by cellsuspension. Add the liposomes loaded with vitamin A in a finalconcentration of 5×10⁻⁶ M. Prepare parallel samples with pure vitamin Aand empty liposomes as controls. For the measurement of MMP-9 and TIMP-1in the culture supernatant and for determination of mRNA expressionincubate the cells in an incubator for three days at 37° C. Afterwardsresuspend the cells carefully and first remove 2×10⁴ cells per PCRlysate for lysis in 200 μl of RNAclean (AGS, Heidelberg, Germany)in anEppendorf reaction vial. Store these PCR lysates at −20° C. untilfurther use. Transfer the remaining cell suspension into an Eppendorfvial and centrifuge for 5 min at 14,000×g. Remove the supernatant,transfer into a fresh vial and store at −80° C. until measurement.

Summary of the samples: 1. BAL cells or PBMCs, respectively without anyaddition 2. BAL cells or PBMCs, respectively + empty liposomes 3. BALcells or PBMCs, respectively + liposomes vitA 5 μM final 4. BAL cells orPBMCs, respectively pure vitA 5 μM finalDeterminations of MMP-9 and TIMP-1 on the Protein level by ELISA

For the measurement of MMP-9 and TIMP-1 in cell culture supernatantscommercially available ELISA kits of Amersham Pharmacia Biotech company(Freiburg, Germany) are used. Specifically, the kits are:

-   Matrix metalloproteinase-9 (MMP-9), human, ELISA system: code RPN    2614-   Tissue inhibitor of metalloproteinases-1 (TIMP-1), human, ELISA    system: code RPN 2611-   Matrix metalloproteinase-9 (MMP-9), human, activity system: code RPN    2630

The ELISAs were always performed exactly according to the manufacturer'sinstructions.

Determinations of mRNA Expression of MMP-9 and TIMP-1 by Means ofSemi-Quantitative RT-PCR

The method used is established and already published in severalpublications. Therefore, only some points are listed for description.

-   -   isolation of total RNA using the RNAclean method    -   transcription of the RNA into cDNA using oligo-dT    -   amplification of the cDNA using specific primers for MMP-9,        TIMP-1, and α-enolase as a control    -   confirmation by agarose gel        Preparation of Liposomes Containing 5×10⁻⁴ M of All-Trans        Tetinoic Acid        Reagents:    -   phosphatidyl choline:        1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholin=POPC    -   phosphatidyl serine: 1,1-dioleyl-sn-glycero-3-phospho-L-serine,        mono sodium salt=OOPS    -   all-trans retinoic acid (vitamin A): Sigma # R-2625        (Deisenhofen, Germany) 10 mM stock solution in absolute ethanol    -   (±)-α-tocopherol (vitamin E): Sigma # T-3251 (Deisenhofen) stock        solution 20 mg/ml in absolute ethanol

Be careful to ensure that all reagents used are endotoxin-free(LPS-free) and render all glass ware LPS-free by baking at 180° C. for 3h. Charge 17.5 mg POPC and 7.5 mg OOPS into a sterile freezing tube(NUNC) to prepare 1 ml liposomes. Dissolve lipids by addition of 1 mlchloroform (Merck). Transfer the complete solution into a 250 ml Duranglass round bottom flask and add 50 μl of the vitamin A stock solutionwith a pipette. Additionally add 10 μl of the vitamin E stock solutionto protect lipids and vitamin A from oxidation. Under sterileconditions, evaporate the chloroform during gentle heating (not morethan 42° C.) and rotation of the flask until a whitish lipid film formsat the inner glass surface. Reconstitute the liposomes by addition of 1ml sterile LPS-free phosphate buffer (PBS) and agitation of the flask.For complete reconstitution of the liposomes let the preparation restfor about 20 min at room temperature (protected from light). Afterwards,transfer the resulting multilamellar liposomes into a sterile 1.5 mlEppendorf reaction vial. For control purposes, always prepare ‘empty’liposomes without vitamin A addition. To remove vitamin A notencapsulated into liposomes centrifuge the liposome preparation in anEppendorf table top centrifuge at 14,000×g for 5 min.

Pipette off the supernatant and restore the volume to 1 ml using freshPBS. Repeat this washing step for a total of 6 times.

In the subsequent extrusion process the multilamellar liposomes areconverted into unilamellar liposomes having a diameter of 0.4 μm. Forthis purpose, the extruder is assembled according to the instructions ofthe manufacturer (Lipex Biomembranes, Vancouver, Canada). To obtain 0.4μm liposomes a filter with a pore size of 0.4 Am (Costar Nuclepore # 110407) is inserted in addition to the preliminary filter (Drain Disc,Costar # 230 300). Charge the complete volume of liposomes into the topof the device using a Pasteur pipette. Extrude liposomes under pressure(about 5–10 bar, not more than 40 bar) and recover in an Eppendorfreaction vial. Recharge these liposomes into the top of the device andrepeat (the membrane filter may be used up to 9 times). A liposomepreparation should be subjected to this cycle at least three times(preferably five times). For use in cell culture, subject the finishedunilamellar liposomes a sterile filtration through Millipore # SLGV 013OS filter in a sterile work bench. A liposome preparation prepared inthis way may be used for at least four weeks.

Characterization of the Liposomes

-   -   unilamellar    -   0.4 μm in diameter    -   confirmation of functionality may be carried out using a        specific staining method and analysis via flow cytometer (may be        described in detail if necessary)

1. A pharmaceutical composition comprising a therapeutically effectiveamount of vitamin A, a vitamin A derivative, or a combination thereofwithin a unilamellar liposome targeted to receptors of alveolarmacrophages, wherein: (a) the lipid composition of said unilamellarliposome consists of phosphatidylcholine and at least one otherphospholipid selected from the group consisting of phosphatidylinositol,phosphatidylserine, and cardiolipin in their lipid bilayer; (b) theratio of said phosphatidylcholine to said at least one otherphospholipid is 75:25 to 65:35; (c) said unilamellar liposome has adiameter of about 0.1 μm to about 0.4 μm; and d) the vitamin Aderivative is selected from the group consisting of retinol, retinal,retinoic acid, β-carotene, and esters thereof, and further wherein thepharmaceutical composition is in an inhalable form for treatment oftracheo-bronchial diseases in a subject.
 2. The pharmaceuticalcomposition according to claim 1, wherein said liposome contains astabilizer.
 3. The pharmaceutical composition according to claim 2,wherein an antioxidant is used as said stabilizer.
 4. The pharmaceuticalcomposition according to claim 2, wherein vitamin E is used as saidstabilizer.
 5. The pharmaceutical composition according to claim 2,wherein said stabilizer is used in an amount of 0.1–10% per 25 mglipids.
 6. The pharmaceutical composition according to claim 1, whereinphosphatidylserine is present in an amount of 25–35% of totalphospholipid.
 7. The pharmaceutical composition according to claim 1,wherein said liposomes contain 5.0 mg to 0.0015 mg of the vitamin A,vitamin A derivative, or the combination thereof per 25 mg lipids. 8.The pharmaceutical composition according to claim 1, wherein saidcomposition further contains carriers and auxiliary agents.
 9. Thepharmaceutical composition according to claim 1, wherein saidcomposition contains other active agents besides vitamin A.
 10. Thepharmaceutical composition according to claim 5, wherein said stabilizeris used in an amount of 0.8% per 25 mg lipids.
 11. The pharmaceuticalcomposition according to claim 7, wherein said liposomes contain 0.05 mgto 0.30 mg of the vitamin A, vitamin A derivative, or the combinationthereof per 25 mg lipids.
 12. The pharmaceutical composition of claim 1,wherein the phosphatidylcholine is1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC).
 13. Thepharmaceutical composition of claim 12, wherein the at least one otherphospholipid is phosphatidylserine.
 14. The pharmaceutical compositionof claim 1, wherein the at least one other phospholipid isphosphatidylserine.
 15. The pharmaceutical composition of claim 1,wherein the esters thereof are selected from the group consisting ofsaturated and unsaturated fatty acid esters of retinol and saturated andunsaturated fatty acid alcohol esters of retinoic acid.